坪井 良樹

Thioredoxin-interacting protein (TXNIP) plays an important role in glucose metabolism, and its expression is regulated by DNA methylation (DNAm). Although the association between TXNIP DNAm and type 2 diabetes mellitus has been demonstrated in studies with a cross-sectional design, prospective studies are needed. We therefore examined the association between TXNIP DNAm levels and longitudinal changes in glycemic traits by conducting a longitudinal study involving 169 subjects who underwent two health checkups in 2015 and 2019. We used a pyrosequencing assay to determine TXNIP DNAm levels in leukocytes (cg19693031). Logistic regression analyses were performed to assess the associations between dichotomized TXNIP DNAm levels and marked increases in glycemic traits. At four years, the TXNIP DNA hypomethylation group had a higher percentage of changes in fasting plasma glucose (FPG) and hemoglobin A1c (HbA1c) compared to those in the hypermethylation group. The adjusted odds ratios for FPG and HbA1c levels were significantly higher in the TXNIP DNA hypomethylation group than in the hypermethylation group. We found that TXNIP DNA hypomethylation at baseline was associated with a marked increase in glycemic traits. Leukocyte TXNIP DNAm status could potentially be used as an early biomarker for impaired glucose homeostasis.

The renal angina index (RAI) is a validated scoring tool for predicting acute kidney injury (AKI). We investigated the efficacy of the RAI in 2436 heterogeneous patients (mean age, 70 years) treated in cardiac intensive care units (CICUs). The RAI was calculated from creatinine and patient condition scores. AKI was diagnosed by the Kidney Disease: Improving Global Outcome criteria. The primary and secondary endpoints were the development of severe AKI and all-cause mortality, respectively. Four hundred thirty-three patients developed AKI, 87 of them severe. In multivariate analyses, the RAI was a significant independent predictor of severe AKI. During the 12-month follow-up period, 210 patients suffered all-cause death. Elevated RAI was independently associated with all-cause mortality, as was NT-proBNP (p < 0.001). The RAI is a potent predictor not only of severe AKI but also of adverse outcomes and substantially improved the 12-month risk stratification of patients hospitalized in CICUs.

AIMS: The developmental origin of health and disease (DOHaD) theory postulates that poor nutrition during fetal life increases the risk of disease later in life. Excessive fructose intake has been associated with obesity, diabetes, and nonalcoholic fatty liver disease, and maternal fructose intake during pregnancy has been shown to affect offspring health. In this study, we investigated the effects of high maternal fructose intake on the liver stem/progenitor cells of offspring. MAIN METHOD: A fructose-based DOHaD model was established using Sprague-Dawley rats. Small hepatocytes (SHs), which play an important role in liver development and regeneration, were isolated from the offspring of dams that were fed a high-fructose corn syrup (HFCS) diet. The gene expression and DNA methylation patterns were analyzed on postnatal day (PD) 21 and 60. KEY FINDINGS: Maternal HFCS intake did not affect body weight or caloric intake, but differences in gene expression and DNA methylation patterns were observed in the SHs of offspring. Functional analysis revealed an association between metabolic processes and ion transport. SIGNIFICANCE: These results suggest that maternal fructose intake affects DNA methylation and gene expression in the liver stem/progenitor cells of offspring. Furthermore, the prolonged retention of these changes in gene expression and DNA methylation in adulthood (PD 60) suggests that maternal fructose intake may exert lifelong effects. These findings provide insights into the DOHaD for liver-related disorders and highlight the importance of maternal nutrition for the health of the next generation.

OBJECTIVES: The mitochondrial DNA (mtDNA) is unique and circular with multiple copies of the genome. The lower mtDNA copy number (mtDNA-CN) in leukocytes is associated with the risk of all-cause mortality. However, its long-term association is unknown. Thus, the study examined the association between mtDNA-CN and the risk of all-cause mortality in a long-term follow-up study in the Japanese population. DESIGN: This longitudinal study included the study cohort from an annual, population-based health checkup in the town of Yakumo, Hokkaido, Japan. SETTING AND PARTICIPANTS: 814 participants (baseline age range: 38-80 years, mean: 56.3 years) were included in this study in 1990. They were followed-up regarding mortality for about 30 years (median: 28.1 years) till 2019. MEASURES: The genomic DNA was extracted from peripheral blood mononuclear cells and the mtDNA-CN was measured using real-time polymerase chain reaction. The level of the mtDNA-CN was divided into tertiles (low, middle, and high). The participants were categorized based on their age into middle-aged (<60 years old) or old-aged (≥60 years old). Survival analysis was performed for tertile of mtDNA-CN and compared using the log-rank test. Univariate and multivariable Cox proportional hazard regression analyses were performed to assess the association between mtDNA-CN and all-cause mortality. The model adjusted with age, sex, body mass index, systolic blood pressure, smoking habit, alcohol consumption, exercise habit, and education level. RESULTS: The low levels of mtDNA-CN resulted in a significant decrease in cumulative survival rate (P <  0.05). The risk of mortality was significantly higher in the middle-aged cohort when mtDNA-CN levels were low (hazard ratios [95% confidence intervals]: 1.98 [1.10-3.56]). CONCLUSION: This study demonstrated that leukocyte mtDNA-CN is associated with future mortality risk. Our study findings may lead to further research on the early prediction of mortality and its underlying mechanisms.

Background: Carotenoids have been reported to exert protective effects against age-related diseases via changes in DNA methylation. Although lower thioredoxin-interacting protein (TXNIP) DNA methylation is associated with age-related diseases, only a few studies have investigated the factors influencing TXNIP DNA methylation. Carotenoids may be a factor linking TXNIP to specific pathophysiological functions. The aim of this study was to examine whether serum carotenoid levels are associated with TXNIP DNA methylation levels. Methods: We conducted a cross-sectional study using 376 health examination participants (169 men). DNA methylation levels were determined using a pyrosequencing assay. Serum carotenoid levels were determined by high-performance liquid chromatography. Multivariable regression analyses were performed to examine the associations between TXNIP DNA methylation levels and serum carotenoid levels with adjustment for age, BMI, HbA1c, CRP, smoking habits, alcohol consumption, exercise habits, and percentage of neutrophils. Results: Multiple linear regression analyses showed that TXNIP DNA methylation levels were positively associated with serum levels of zeaxanthin/lutein (β [95%CI]: 1.935 [0.184, 3.685]), β-cryptoxanthin (1.447 [0.324, 2.570]), α-carotene (1.061 [0.044, 2.077]), β-carotene (1.272 [0.319, 2.226]), total carotenes (1.255 [0.040, 2.469]), total xanthophylls (2.133 [0.315, 3.951]), provitamin A (1.460 [0.402, 2.519]), and total carotenoids (1.972 [0.261, 3.683]) in men (all p<0.05). Of these, provitamin A showed the stronger association (standardized β=0.216). No significant association of TXNIP DNA methylation and serum carotenoid was observed in women. Conclusions: The findings of this study suggest that carotenoid intake may protect against age-related diseases by altering TXNIP DNA methylation status in men.

BACKGROUND: Epigenetic studies have reported relationships between dietary nutrient intake and methylation levels. However, genetic variants that may affect DNA methylation (DNAm) pattern, called methylation quantitative loci (mQTL), are usually overlooked in these analyses. We investigated whether mQTL change the relationship between dietary nutrient intake and leukocyte DNAm levels with an example of estimated fatty acid intake and ATP-binding cassette transporter A1 (ABCA1). METHODS: A cross-sectional study on 231 participants (108 men, mean age: 62.7 y) without clinical history of cancer and no prescriptions for dyslipidemia. We measured leukocyte DNAm levels of 8 CpG sites within ABCA1 gene by pyrosequencing method and used mean methylation levels for statistical analysis. TaqMan assay was used for genotyping a genetic variant of ABCA1 (rs1800976). Dietary fatty acid intake was estimated with a validated food frequency questionnaire and adjusted for total energy intake by using residual methods. RESULTS: Mean ABCA1 DNAm levels were 5% lower with the number of minor alleles in rs1800976 (CC, 40.6%; CG, 35.9%; GG, 30.6%). Higher dietary n-3 PUFA intake was associated with lower ABCA1 DNAm levels (1st (ref) vs. 4th, β [95% CI]: -2.52 [-4.77, -0.28]). After controlling for rs180076, the association between dietary n-3 PUFA intake and ABCA1 DNAm levels was attenuated, but still showed an independent association (1st (ref) vs. 4th, β [95% CI]: -2.00 [-3.84, -0.18]). The interaction of mQTL and dietary n-3 PUFA intake on DNAm levels was not significant. CONCLUSIONS: This result suggested that dietary n-3 PUFA intake would be an independent predictor of DNAm levels in ABCA1 gene after adjusting for individual genetic background. Considering mQTL need to broaden into other genes and nutrients for deeper understanding of DNA methylation, which can contribute to personalized nutritional intervention.

Background: The increasing prevalence of non-alcoholic fatty liver disease (NAFLD) has become a global health problem. NAFLD has few initial symptoms and may be difficult to detect early, so there is need for a minimally invasive early detection marker. We hypothesized that miR-122 and miR-20a levels combined, as the miR-122/miR-20a ratio might detect NAFLD more sensitively. Methods: This study involved 167 participants with low alcohol intake. Those who had an increase in echogenicity of the liver parenchyma and hepato-renal contrast on ultrasonography were classified as the NAFLD group (n = 44), which was further classified into mild (n = 26) and severe (n = 18) groups based on echogenic intensity and hepatic vessel and diaphragm visualization. Participants without fatty liver were included in the normal group, except for those with an abnormal body mass index, glycated hemoglobin, and systolic blood pressure (n = 123) values. Serum miR-122 and miR-20a expression levels in participants were measured by real-time polymerase chain reaction, and the miR-122/miR-20a was calculated. Results: In the NAFLD group, miR-122 expression was significantly higher and the miR-20a was significantly lower than in the normal group, in agreement with previous studies. miR-122/miR-20a was also significantly higher in the NAFLD group. Receiver operating characteristic curve analysis was performed with miR-122/miR-20a as an NAFLD detection marker, and the area under the curve of miR-122/miR-20a was significantly larger than that of miR-122 or miR-20a alone. Conclusions: The miR-122/miR-20a ratio, combined with miR-122 and miR-20a levels, is a useful biomarker to detect NAFLD with high sensitivity.

We previously reported that maternal fructose consumption increases blood corticosterone levels in rat offspring. However, the underlying mechanism of action remains unclear. In the present study, we aimed to elucidate the molecular mechanism by which maternal high-fructose corn syrup (HFCS) intake increases circulating GC levels in rat offspring (GC; corticosterone in rodents and cortisol in humans). Female Sprague Dawley rats received HFCS solution during gestation and lactation. The male offspring were fed distilled water from weaning to 60 days of age. We investigated the activities of GC-metabolizing enzymes (11β-Hsd1 and 11β-Hsd2) in various tissues (i.e., liver, kidney, adrenal glands, muscle, and white adipose tissue) and epigenetic modification. 11β-Hsd2 activity decreased in the kidney of the HFCS-fed dams. Moreover, the epigenetic analysis suggested that miR-27a reduced Hsd11b2 mRNA expression in the kidney of offspring. Maternal HFCS-induced elevation of circulating GC levels in offspring may be explained by a decrease in 11β-Hsd2 activity via renal miR-27a expression. The present study may allow us to determine one of the mechanisms of GC elevation in rat offspring that is often observed in the developmental origins of the health and disease (DOHaD) phenomenon.

BACKGROUND: Previous studies have proposed different formulas of estimating glomerular filtration rate (eGFR) among clinical patients. The comprehensive comparison of eGFR formulas is not well established in a Japanese population. We compared eGFR values and chronic kidney disease (CKD) classification of nine different eGFR in a Japanese general population sample. METHODS: We analyzed 469 Japanese community-dwelling adults (184 men) without any self-reported kidney disease. GFR estimated using the 4- and 6-parameter Modification of Diet in Renal Disease (MDRD) formulas (MDRD4 and MDRD6); the CKD-EPI formulas based on creatinine with (CKD-EPI-2009) and without race coefficient (CKD-EPI-2021), on cystatin C (CKD-EPI-Cys), on both (CKD-EPI-CreCys); the Japanese creatinine-based formula (JPN-Cre), cystatin C-based formula (JPN-Cys), and modified CKD-EPI formula (JPN-CKD-EPI). CKD stages were defined by KDIGO guidelines (eGFR < 60 ml/min/1.73 m2). RESULTS: eGFRJPN-Cre (mean = 71.2; SD = 14.3) were much lower than eGFRCKD-EPI-2021 (mean = 94.2; SD = 12.7), while eGFRJPN-Cys (mean = 102.8; SD = 24.2) was comparable to the MDRD and CKD-EPI formulas. The difference between eGFRCKD-EPI-2021 and eGFRJPN-Cre showed a V-shaped distribution across eGFR levels, indicating complex errors between these formulas. We observed very low agreement in CKD classification between eGFRJPN-Cre and the eGFRCKD-EPI-2021 (kappa = 0.13; 95% confidence interval: 0.06, 0.23). CONCLUSIONS: JPN-Cre was substantially different from the CKD-EPI formula without race term (CKD-EPI-2021), which means that it is impossible to recalibrate those with a simple coefficient. Although a comparison with measured GFR should be necessary, choice of the estimation method needs caution in clinical decision-making and academic research.

DNA methylation can be affected by numerous lifestyle factors, including diet. Tobacco smoking induces aryl hydrocarbon receptor repressor (AHRR) DNA hypomethylation, which increases the risk of lung and other cancers. However, no lifestyle habits that might increase or restore percentage of AHRR DNA methylation have been identified. We hypothesized that dietary intakes of vegetables/fruits and serum carotenoid concentrations are related to AHRR DNA methylation. A total of 813 individuals participated in this cross-sectional study. A food frequency questionnaire was used to assess dietary intake of vegetables and fruits. AHRR DNA methylation in peripheral blood mononuclear cells were measured using pyrosequencing method. In men, dietary fruit intake was significantly and positively associated with AHRR DNA methylation among current smokers (P for trend = .034). A significant positive association of serum provitamin A with AHRR DNA methylation was observed among current smokers (men: standardized β = 0.141 [0.045 to 0.237], women: standardized β = 0.570 [0.153 to 0.990]). However, compared with never smokers with low provitamin A concentrations, percentages of AHRR DNA methylation were much lower among current smokers, even those with high provitamin A concentrations (men: β = -19.1% [-33.8 to -19.8], women: β = -6.0% [-10.2 to -1.7]). Dietary intake of vegetables and fruits rich in provitamin A may increase percentage of AHRR DNA methylation in current smokers. However, although we found a beneficial effect of provitamin A on AHRR DNA methylation, this beneficial effect could not completely remove the effect of smoking on AHRR DNA demethylation.

Carotenoids are abundant pigments mainly contained in vegetables and fruits, and show antioxidant properties by quenching free radicals in human body. Few studies have investigated associations between serum carotenoid levels and premature mortality. The objective of this study was to investigate the association between serum carotenoid level and premature mortality in a Japanese population. This study included 446 Japanese adults (174 men, aged of 40-64) recruited as participants in the Japan Collaborative Cohort (JACC) Study. Serum carotenoid level was measured by high-performance liquid chromatography. Premature mortality was defined as death before 65 years old during the follow-up period. Premature mortality was ascertained in 60 men (34.5%) and 65 women (23.9%). In men, compared to the 1st tertile of serum β-cryptoxanthin and provitamin A, those who were in the 3rd tertile had lower risks of premature all-cause mortality (OR, 95% CI: 0.19, 0.07-0.47 for β-cryptoxanthin, and 0.24, 0.09-0.61 for provitamin A). In women, compared to the 1st tertile of serum β-cryptoxanthin, those who were in the 3rd tertile had higher risks of premature all-cause mortality (OR, 95% CI: 1.94, 1.00-4.03). These significant associations were observed in analyses for premature cancer mortality. We found significant associations between higher levels of serum β-cryptoxanthin and provitamin A and lower risks of premature mortality among Japanese men, while a different directional association was found in women. Although these findings suggest roles of serum carotenoids on premature mortality, further studies are needed to validate this association in other populations.

BACKGROUND: Although many polygenic risk scores (PRS) for cardiovascular traits have been developed in European populations, it is an urgent task to construct a PRS and to evaluate its ability in non-European populations. We developed a genome-wide PRS for blood pressure in a Japanese population and examined the associations between this PRS and hypertension prevalence. METHODS: We performed a cross-sectional study in 11 252 Japanese individuals who participated in the J-MICC (Japan Multi-Institutional Collaborative Cohort) study. Using publicly available GWAS summary statistics from Biobank Japan, we developed the PRS in the target data (n=7876). With >30 000 single nucleotide polymorphisms, we evaluated PRS performance in the test data (n=3376). Hypertension was defined as systolic blood pressure of 130 mm Hg or more, or diastolic blood pressure of 85 mm Hg or more, or taking an antihypertensive drug. RESULTS: Compared with the middle PRS quintile, the prevalence of hypertension at the top PRS quintile was higher independently from traditional risk factors (odds ratio, 1.73 [95% CI, 1.32-2.27]). The difference of mean systolic blood pressure and diastolic blood pressure between the middle and the top PRS quintile was 4.55 (95% CI, 2.26-6.85) and 2.32 (95% CI, 0.86-3.78) mm Hg, respectively. Subgroups reflecting combinations of Japanese PRS and modifiable lifestyles and factors (smoking, alcohol intake, sedentary time, and obesity) were associated with the prevalence of hypertension. A European-derived PRS was not associated with hypertension in our participants. CONCLUSIONS: A PRS for blood pressure was significantly associated with hypertension and BP traits in a general Japanese population. Our findings also highlighted the importance of a combination of PRS and risk factors for identifying high-risk subgroups.

BACKGROUND: Smoking is well known to be a major risk factor for cancer, and to decrease the levels of aryl hydrocarbon receptor repressor (AHRR) DNA methylation. AHRR is a key regulator for AHR signaling, which is involved in chemical metabolism and cancer development. Therefore, smoking-induced AHRR DNA hypomethylation may be associated with cancer development. However, it has not been reported that association between AHHR DNA methylation and cancer mortality in Asian population. Hence, we examined whether AHRR DNA methylation levels were associated with cancer mortality in a Japanese population. METHODS: This study was conducted with 812 participants (aged 38-80 years) who received a health check-up in 1990, and did not have a clinical histories. We followed up the participants until the end of 2019 (median: 27.8 years), and 100 participants died from cancer. The AHRR DNA methylation levels in peripheral blood mononuclear cells (PBMCs) were measured by the pyrosequencing method. We calculated the hazard ratio (HR) and 95% confidence interval (CI) for cancer mortality according to the baseline levels of AHRR DNA methylation. RESULTS: We found that AHRR DNA hypomethylation was associated with a higher risk of all cancer mortality, especially smoking related cancers and lung cancer. (all cancer: HR, 1.28, 95% CI, 1.09-1.51; smoking-related cancers: HR, 1.35, 95% CI, 1.12-1.62; lung cancer: HR, 1.68, 95% CI, 1.24-2.26). CONCLUSIONS: Smoking-induced AHRR DNA hypomethylation in PBMCs was associated with the risk of cancer mortality in Japanese population; therefore, hypomethylation of AHRR may be a useful biomarker of cancer mortality risk.

Background: Thioredoxin-interacting protein (TXNIP) controls the cellular redox balance by binding to and inhibiting the expression and function of thioredoxin. DNA methylation of the TXNIP gene is involved in the regulation of TXNIP mRNA expression. Changes in TXNIP DNA methylation levels are associated with the development of various diseases such as type 2 diabetes mellitus (T2DM). However, few studies have focused on the influence of lifestyle factors such as alcohol intake on TXNIP DNA methylation.Objectives: This research examines the association of drinking behaviors with TXNIP DNA methylation levels in the general Japanese population.Methods: We conducted a cross-sectional study of 404 subjects (176 males and 228 females) who were divided into non-, moderate and heavy drinkers based on self-reported drinking behaviors. TXNIP DNA methylation levels in leukocytes were determined using a pyrosequencing assay.Results: The mean TXNIP DNA methylation level in heavy drinkers (74.2%) was significantly lower than that in non- and moderate drinkers (non: 77.7%, p < .001; moderate: 76.6%, p = .011). Multivariable linear regression analysis showed that log-transformed values of daily (b = -1.34; p < .001) and cumulative (b = -1.06; p = .001) alcohol consumption were associated with decreased TXNIP DNA methylation levels.Conclusion: TXNIP DNA methylation levels in heavy drinkers was lower than in non- and- moderate drinkers. Decreased TXNIP DNA methylation level increases the expression of TXNIP and elevates the risk of developing of diseases such as T2DM. Therefore, decreasing alcohol use in heavy drinkers may lessen the likelihood of some alcohol-related illnesses moderated through TXNIP DNA methylation.

The increasing prevalence of nonalcoholic fatty liver disease (NAFLD) is a global health problem. In recent years, the inhibitory effect of brain-derived neurotrophic factor (BDNF) on diabetes mellitus and fatty liver has been clarified. The purpose of this study was to analyze the relationship between serum BDNF and NAFLD which caused by abnormal metabolism of glucose and lipids. This cross-sectional study involved 429 participants (mean age, 63.5 years: men, 38.5%) with low alcohol intake. Of the participants, those who had an increase in echogenicity of the liver parenchyma and hepato-renal contrast on ultrasonography were classified as the NAFLD group (n = 88), and the others were classified as the normal (n = 341) group. The NAFLD group was further classified into a mild group (n = 60) and a severe group (n = 28) based on the intensity of echogenicity and visualization of the hepatic vessels and diaphragm. Median BDNF levels were higher in the NAFLD group than the normal group (35.5 vs. 42.3 ng/mL, p < 0.01). Furthermore, BDNF levels tended to be associated with the severity of NAFLD (p < 0.01). In addition to the univariate analysis, in the sex- and age-adjusted model, there was a significant association between the BDNF levels and NAFLD severity (p < 0.01). The fully adjusted regression analysis also showed a positive association between the serum BDNF level and NAFLD (p < 0.01). These results suggest that NAFLD patients have a compensatory increase in circulating BDNF levels.

Metabolic syndrome (MetS) is cluster of metabolic diseases, including abdominal obesity, hyperglycemia, high blood pressure, and dyslipidemia, that directly escalate the risk of type 2 diabetes, heart disease, and stroke. Thioredoxin-interacting protein (TXNIP) is a binding protein for thioredoxin, a molecule that is a key inhibitor of cellular oxidation, and thus regulates the cellular redox state. Epigenetic alteration of the TXNIP-encoding locus has been associated with components of MetS. In the present study, we sought to determine whether the level of TXNIP methylation in blood is associated with MetS in the general Japanese population. DNA was extracted from the peripheral blood cells of 37 subjects with and 392 subjects without MetS. The level of TXNIP methylation at cg19693031 was assessed by the bisulfite-pyrosequencing method. We observed that TXNIP methylation levels were lower in MetS subjects (median 74.9%, range 71.7-78.4%) than in non-MetS subjects (median 77.7%, range 74.4-80.5%; p = 0.0024). Calculation of the confounding factor-adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for hypomethylation revealed that subjects with MetS exhibited significantly higher ORs for hypomethylation than did those without MetS (OR, 2.92; 95% CI, 1.33-6.62; p = 0.009). Our findings indicated that lower levels of TXNIP methylation are associated with MetS in the general Japanese population. Altered levels of DNA methylation in TXNIP at cg19693031 might play an important role in the pathogenesis of MetS.

BACKGROUND: To evaluate the utility of histogram analysis (HA) of apparent diffusion coefficient (ADC) values to predict the overall survival (OS) in patients with pancreatic ductal adenocarcinoma (PDAC) and to correlate with pathologically evaluated massive intratumoral necrosis (MITN). MATERIALS AND METHODS: Thirty-nine patients were included in this retrospective study with surgically resected PDAC who underwent preoperative magnetic resonance imaging. Twelve patients received neoadjuvant chemotherapy. HA on the ADC maps were performed to obtain the tumor HA parameters. Using Cox proportional regression analysis adjusted for age, time-dependent receiver-operating-characteristic (ROC) curve analysis, and Kaplan-Meier estimation, we evaluated the association between HA parameters and OS. The association between prognostic factors and pathologically confirmed MITN was assessed by logistic regression analysis. RESULTS: The median OS was 19.9 months. The kurtosis (P < 0.001), entropy (P = 0.013), and energy (P = 0.04) were significantly associated with OS. The kurtosis had the highest area under the ROC curve (AUC) for predicting 3-year survival (AUC 0.824) among these three parameters. Between the kurtosis and MITN, the logistic regression model revealed a positive correlation (P = 0.045). Lower survival rates occurred in patients with high kurtosis (cutoff value > 2.45) than those with low kurtosis (≤ 2.45) (P < 0.001: 1-year survival rate, 75.2% versus 100%: 3-year survival rate, 14.7% versus 100%). CONCLUSIONS: HA derived kurtosis obtained from tumor ADC maps might be a potential imaging biomarker for predicting the presence of MITN and OS in patients with PDAC.

BACKGROUND: Although a number of microRNAs (miRNA) reflecting kidney function has been identified, prospective studies are now urgently needed to determine a clinical utility of these miRNAs among general populations. The purpose of this study was to examine the associations between serum miRNAs and kidney function in a population-based study. METHODS: We conducted a five-year prospective study (2012-2017) of 169 individuals without chronic kidney disease (CKD) at the baseline survey (mean age, 62.5; 96 women). The real-time qPCR was used to measure serum levels of five previously reported miRNAs. Participants with eGFR < 60 mL/min/1.73 m2 were defined as having CKD. Changes in eGFR were defined as eGFR2017 - eGFR2012. RESULTS: After adjusting for covariates including baseline eGFR, lower serum levels (1st tertile) of miR-126 were associated with a greater decline of eGFR (β [SE] = -3.18 [1.50]) and a higher odds ratio (OR) of CKD onset over five years (OR [95% CI] = 3.85 [1.01-16.8]), compared with the 3rd tertile. CONCLUSIONS: We found baseline serum miR-126 levels were associated with changes in eGFR and new CKD cases in a five-year prospective study. This result suggests that miR-126 may be a potential biomarker of CKD even among general populations.

DNA methylation (DNAm) is one of the most studied epigenetic modifications. DNAm has emerged as a key biological mechanism and biomarkers to test associations between environmental exposure and outcomes in epidemiological studies. Although previous studies have focused on associations between DNAm and either exposure/outcomes, it is useful to test for mediation of the association between exposure and outcome by DNAm. The purpose of this scoping review is to introduce the methodological essence of statistical mediation analysis and to examine emerging epidemiological research applying mediation analyses. We conducted this scoping review for published peer-reviewed journals on this topic using online databases (PubMed, Scopus, Cochrane, and CINAHL) ending in December 2020. We extracted a total of 219 articles by initial screening. After reviewing titles, abstracts, and full texts, a total of 69 articles were eligible for this review. The breakdown of studies assigned to each category was 13 for smoking (18.8%), 8 for dietary intake and famine (11.6%), 6 for other lifestyle factors (8.7%), 8 for clinical endpoints (11.6%), 22 for environmental chemical exposures (31.9%), 2 for socioeconomic status (SES) (2.9%), and 10 for genetic factors and race (14.5%). In this review, we provide an exposure-wide summary for the mediation analysis using DNAm levels. However, we found heterogenous methods and interpretations in mediation analysis with typical issues such as different cell compositions and tissue-specificity. Further accumulation of evidence with diverse exposures, populations and with rigorous methodology will be expected to provide further insight in the role of DNAm in disease susceptibility.

OBJECTIVES: To determine the optimal scan delay corresponding to individual hemodynamic status for pancreatic parenchymal phase in dynamic contrast-enhanced CT of the abdomen. METHODS: One hundred and fourteen patients were included in this retrospective study (69 males and 45 females; mean age, 67.9 ± 12.1 years; range, 39-87 years). These patients underwent abdominal dynamic contrast-enhanced CT between November 2019 and May 2020. We calculated and recorded the time from contrast material injection to the bolus-tracking trigger of 100 Hounsfield unit (HU) at the abdominal aorta (s) (TimeTRIG) and scan delay from the bolus-tracking trigger to the initiation of pancreatic parenchymal phase scanning (s) (TimeSD). The scan delay ratio (SDR) was defined by dividing the TimeSD by TimeTRIG. Non-linear regression analysis was conducted to assess the association between CT number of the pancreas and SDR and to reveal the optimal SDR, which was ≥120 HU in pancreatic parenchyma. RESULTS: The non-linear regression analysis showed a significant association between CT number of the pancreas and the SDR (p < 0.001). The mean TimeTRIG and TimeSD were 16.1 s and 16.8 s, respectively. The SDR to peak enhancement of the pancreas (123.5 HU) was 1.00. An SDR between 0.89 and 1.18 shows an appropriate enhancement of the pancreas (≥120 HU). CONCLUSION: The CT number of the pancreas peaked at an SDR of 1.00, which means TimeSD should be approximately the same as TimeTRIG to obtain appropriate pancreatic parenchymal phase images in dynamic contrast-enhanced CT with bolus-tracking method. ADVANCES IN KNOWLEDGE: The hemodynamic state is different in each patient; therefore, scan delay from the bolus-tracking trigger should also vary based on the time from contrast material injection to the bolus-tracking trigger. This is necessary to obtain appropriate late hepatic arterial or pancreatic parenchymal phase images in dynamic contrast-enhanced CT of the abdomen.

Importance: The associations of levels of diverse serum carotenoids ascertained via repeated measurements with all-cause, cancer, and cardiovascular disease (CVD) mortality risk have not been considered in previous prospective studies. Objective: To investigate the association between repeated measurement of serum carotenoid levels and all-cause and cause-specific mortality risk. Design, Setting, and Participants: This cohort study's baseline data were collected using information from physical examinations from 1990 to 1999. Eligible participants were followed up until December 2017, with a median (interquartile range) follow-up period of 22.3 (15.5-25.3) years. Included individuals were age 40 years or older at the baseline data collection, were residents of the study site in the town of Yakumo, Hokkaido, Japan, and participated in a physical examination at least once from 1990 to 1999. Among eligible participants, after excluding 332 individuals, 3116 individuals were included in the analysis. Data analysis was conducted in April 2020. Exposures: Repeated measurements of 6 serum carotenoid levels and 4 associated indices. Main Outcomes and Measures: All-cause, cancer, and CVD mortality, categorized by International Classification of Diseases, Ninth Revision and International Statistical Classification of Diseases and Related Health Problems, Tenth Revision codes, were recorded. A time-dependent Cox regression model was performed to examine associations between time-varying serum carotenoid levels and mortality. Results: Among 3116 individuals who received physical examinations, the mean (SD) age was 54.7 (10.6) years and 1883 (60.4%) were women. During the follow-up period, 762 deaths from all causes, 253 deaths from cancer, and 210 deaths from CVD were ascertained. In a time-dependent Cox regression analysis, for every 25% higher serum levels of total carotenoids, risks were statistically significantly lower for all-cause mortality (hazard ratio [HR], 0.85; 95% CI, 0.82-0.87; P < .001), cancer mortality (HR, 0.82; 95% CI, 0.78-0.87; P < .001), and CVD mortality (HR, 0.86; 95% CI, 0.81-0.91; P < .001). Using only baseline measures, for every 25% higher serum levels of total carotenoids, risks were also statistically significantly lower for all-cause mortality (HR, 0.92; 95% CI, 0.89-0.95; P < .001), cancer mortality (HR, 0.87; 95% CI, 0.83-0.93; P < .001), and CVD mortality (HR, 0.93; 95% CI, 0.88-0.99; P = .03) but with larger HRs than those associated with repeated measurements. Conclusions and Relevance: This study found that higher levels of serum carotenoids in analysis using repeated measurements were associated with significantly lower all-cause and cause-specific mortality over a follow-up period of 25 years.

DNA methylation plays an important role in the pathogenesis and progression of cardiovascular disease (CVD) but the prospective association of DNA methylation with CVD has not been evaluated. Here, we conducted a prospective study to examine whether long interspersed nuclear element-1 (LINE-1) DNA methylation is associated with CVD mortality in a Japanese population. We targeted 822 Japanese who participated in a health check-up in 1990 and had no clinical history of cancer, stroke or ischaemic heart disease. DNA was extracted from peripheral blood mononuclear cells and LINE-1 DNA methylation at three CpG sites was measured using a pyrosequencing method. We used propensity score (PS) matching to reduce the effect of potential confounding. During 18 118.7 persons-years of follow-up, there were 329 deaths from all-causes and 85 deaths from CVD. In PS-matched analysis, a significantly higher HR for CVD mortality was observed in the hypermethylation group than in the hypomethylation group for elderly participants (HR 2.77; 95% CI 1.55 to 4.93). No significant association between LINE-1 DNA methylation and CVD was observed for middle-aged participants. Based on this prospective study, we suggest that LINE-1 DNA hypermethylation is associated with increased CVD mortality risk in an elderly population.

BACKGROUND/AIM: We evaluated the efficacy of primary prophylaxis with pegfilgrastim (PEG) for febrile neutropenia (FN) in small cell lung cancer (SCLC) patients receiving amrubicin (AMR). PATIENTS AND METHODS: A retrospective cohort study was conducted in patients with SCLC receiving AMR as second-line therapy. RESULTS: A total of 33 patients were treated with AMR (no PEG group), while 13 patients were treated with AMR plus prophylactic administration of PEG (PEG group). The severity of neutropenia was significantly reduced in the PEG group compared to the no PEG group (p=0.02). The incidence of FN in the no PEG and PEG groups was 27.3% and 7.7%, respectively. The time to development of FN tended to be longer in the PEG group compared to the no PEG group (p=0.132). CONCLUSION: Primary prophylaxis with PEG may be beneficial in reducing the risk of FN in patients with SCLC receiving AMR.

Objectives: A diet rich in fish and ω-3 polyunsaturated fatty acids (PUFAs) has been thought to reduce the risk for cardiovascular disease (CVD). The beneficial effects of fish oil and ω-3 PUFA on CVD can be mediated by epigenetic status of the genes associated with lipid metabolism and inflammation. The aim of this study was to investigate whether dietary fish and fatty acid (FA) intakes are associated with leukocyte ATP-binding cassette transporter A1 (ABCA1) DNA methylation levels in a Japanese population. Methods: This cross-sectional study included 298 adults (137 men and 161 women) without clinical history of CVD or cancer. The pyrosequencing method was used to measure leukocyte ABCA1 DNA methylation levels. Dietary fish and FA intakes were assessed based on the validated food frequency questionnaire. Results: Mean ABCA1 DNA methylation levels were significantly lower in the highest fish intake groups (≥5–6/wk) compared with the lowest intake group (≤1–2/wk; P = 0.004). In multivariable linear regression analyses, higher dietary intake of ω-3 PUFAs and ω-3 highly unsaturated fatty acids was significantly associated with decreased levels of ABCA1 DNA methylation (P = 0.001 and 0.005); whereas no significant associations were seen between intake of dietary saturated fatty acid, monounsaturated fatty acid, and ω-6 PUFAs and ABCA1 DNA methylation. Conclusion: Higher dietary fish and ω-3 PUFA intake were associated with lower ABCA1 DNA levels in a Japanese population. The present results may bring potential insights on biological mechanisms underlying the protective effects of dietary fish and ω-3 PUFA intakes on CVD.

Thioredoxin-interacting protein (TXNIP) inhibits the activity of thioredoxin (TXN), leading to increased oxidative stress. Expression of the TXNIP gene is regulated by DNA methylation. However, no study has reported the influence of lifestyle factors on TXNIP DNA methylation. Our goal was to determine the association between smoking habits and TXNIP DNA methylation levels in a Japanese population. We conducted a cross-sectional study of 417 subjects (180 males and 237 females) participating in a health examination. We used a pyrosequencing assay to determine TXNIP DNA methylation levels in leukocytes. The mean TXNIP DNA methylation level in current smokers (75.3%) was significantly lower than that in never and ex-smokers (never: 78.1%, p < 0.001; ex: 76.9%, p = 0.013). Multivariable logistic regression analyses showed that the OR for TXNIP DNA hypomethylation was significantly higher in current smokers than that in never smokers, and significantly higher in current smokers with years of smoking ≥ 35 and Brinkman Index ≥ 600 compared to that in nonsmokers. In conclusion, we found that current smokers had TXNIP DNA hypomethylation compared to never and ex-smokers. Moreover, long-term smoking and high smoking exposure also were associated with TXNIP DNA hypomethylation.

BACKGROUND: Higher intake of fruits and vegetables is associated with reduced risk of specific types of cancer and of cardiovascular disease (CVD), but the protective role of the vitamins contained in fruits and vegetables on CVD is controversial. This discrepancy can raise the question of the effects of antioxidants in vitamins on CVD. Recently, we reported that higher vegetable intake was significantly associated with the decreased DNA methylation level of ATP-binding cassette transporter A1 (ABCA1), a gene associated with HDL-cholesterol metabolism. OBJECTIVE: We investigated whether ABCA1 DNA methylation mediates an effect of dietary vitamin intake on lipid profiles, an important risk factor for CVD, in a Japanese population. METHODS: A total of 225 individuals (108 men and 117 women) with no clinical history and no drug use for dyslipidemia participated in this cross-sectional study. We used the pyrosequencing method to measure the ABCA1 DNA methylation levels at 8 CpG sites, and we used mean DNA methylation level in statistical analysis. Dietary vitamin intake was assessed with the FFQ and adjusted for the residual method. RESULTS: In women, higher dietary vitamin intake [vitamin A, β-carotene, folic acid, vitamin C (VC), vitamin D, and vitamin E] was significantly associated with lower mean ABCA1 DNA methylation levels (P = 0.004, 0.03, 0.005, 0.001, 0.03, and 0.04, respectively). In addition, in women, we found a significant inverse association between mean ABCA1 DNA methylation and HDL cholesterol (P = 0.04) but not for other lipid indexes. Mediation analysis showed a significant indirect effect of VC intake on HDL cholesterol through ABCA1 DNA methylation level in women (P = 0.04). CONCLUSIONS: Although this study does not prove causality, the results suggest that ABCA1 DNA methylation mediates the protective effect of VC on HDL cholesterol in women, which could offer a novel biological mechanism in CVD prevention.

Objectives: MicroRNAs (miRNAs) dysregulate gene expression by binding to target messenger RNAs, and play an important role in the pathogenesis of various diseases, including cancers, cardiovascular diseases and diabetes. Circulating miRNAs have increasingly been recognized as biomarkers for detecting and diagnosing those diseases. Few studies have investigated the association of circulating miRNA with the early stages of cognitive impairment, such as mild cognitive impairment, in the general population. The purpose of this study was to examine the association between cognitive function and several serum miRNAs levels related to amyloid precursor protein (APP) proteolysis in a Japanese general population who had never been diagnosed with dementia.Methods: We conducted a cross-sectional study of 337 Japanese subjects (144 men, 193 women) who attended a health examination. The short form of the Mini-Mental State Examination (SMMSE) was used to assess cognitive function. Serum levels of 6 miRNAs (let-7d, miR-17, miR-20a, miR-27a, miR-34a, miR-103a) were measured by quantitative real-time polymerase chain reaction.Results: Multivariable-adjusted odds ratios (ORs) for lower SMMSE score (SMMSE score < 28) were significantly increased in the lowest tertile of serum miR-20a (OR, 2.08; 95% confidence interval (CI), 1.09-4.04) and miR103a (OR, 1.91; 95% CI, 1.00-3.69) compared to the highest tertile. Moreover, serum levels of miR-20a, -27a, and -103a were linearly and positively associated with SMMSE scores after adjustment for confounding factors.Conclusion: Low serum levels of miR-20a, -27a, and -103a are independently associated with cognitive impairment.

OBJECTIVE: Dietary intake of vegetables is one of the key lifestyle factors associated with preventing cancer and cardiovascular disease (CVD). Although previous studies have provided evidence that dietary factors can alter global DNA methylation levels in humans, little work has been done on dietary factors influencing methylation levels of specific genes associated with CVD. The aim of this study was to examine whether dietary intake of vegetables was associated with adenosine triphosphate-binding membrane cassette transporter A1 (ABCA1) DNA methylation levels in leukocytes in a Japanese population. METHODS: This cross-sectional study included 279 Japanese adults (125 men, 154 women) without any clinical history of cancer, stroke, or ischemic heart disease. ABCA1 DNA methylation levels in leukocytes were measured using a pyrosequencing method. Information on dietary vegetable intake was obtained from the validated food frequency questionnaire. RESULTS: Mean ABCA1 DNA methylation levels in men and women were 35.6% ± 6.5% and 36.9% ± 6.7%, respectively. In women, multivariable linear regression analysis showed that the group with the highest dietary vegetable intake (carrot, broccoli, pumpkin, and all vegetables) showed significantly lower levels of ABCA1 DNA methylation than the lowest intake group (P = 0.04, <0.001, 0.001, and 0.02, respectively). No significant association was observed between dietary intake of vegetables and DNA methylation levels in men. CONCLUSIONS: High dietary intake of vegetables was associated with decreased ABCA1 DNA methylation levels in Japanese women. This may contribute to a better understanding of the protective effects of dietary vegetable intake on CVD.

Purpose It has been demonstrated that circulating microRNA profiles are affected by physiological conditions. Several studies have demonstrated that microRNAs play important roles in the regulation of adiposity. However, few have investigated the relationship between circulating microRNAs and obesity, which has become a major public health problem worldwide. This study investigated the association between circulating microRNAs and obesity in a Japanese population. Methods Obesity parameters, such as subcutaneous and visceral fat adipose tissue, body fat percentage, and body mass index were assessed in a cross-sectional sample of 526 participants who attended health examinations in Yakumo, Japan. In addition, five circulating microRNAs (miR-20a, -21, -27a, -103a, and -320), which are involved in adipocyte proliferation and differentiation, were quantified using real-time polymerase chain reaction amplification. Results We compared the circulating microRNA concentrations in a percentile greater than 75th (high) with below the value (low) of subcutaneous adipose tissue, visceral fat adipose tissue, body mass index, and per cent body fat. For visceral fat adipose tissue, significant decrease in miR-320 expression was observed in high group. Also, for body mass index, significant change of miR-20a, -27a, 103a, and 320 expression level was observed in high group. Multiple linear regression analysis demonstrated that circulating levels of some microRNA such as miR-27a were significantly associated with subcutaneous adipose tissue, visceral fat adipose tissue, and body mass index. Conclusions Our findings support the need for further studies to determine whether such changes are consistent across different populations and whether the identified microRNAs may represent novel biomarkers to predict the susceptibility and progression of obesity-related disorders.

AIM: Aberrant global DNA methylation is involved in the development of several diseases, including cardiovascular disease (CVD). We investigated whether the methylation of long interspersed nuclear element-1 (LINE-1) in leukocytes is associated with dyslipidemia, a major risk factor for CVD, in the Japanese general population. METHODS: We conducted a cross-sectional study consisting of 420 Japanese subjects (187 men and 233 women) without a clinical history of cancer, stroke, or ischemic heart disease. LINE-1 DNA methylation levels in leukocytes were measured using a pyrosequencing method. RESULTS: Significantly higher odds ratios (ORs) for hypermethylation were observed in the high LDL cholesterol and high LDL/HDL ratio groups than the corresponding normal group (high LDLC group: OR, 1.88; 95% confidence interval [CI], 1.20-2.96, high LDL/HDL ratio group: OR, 1.90; 95% CI, 1.20-3.01). Subjects with 2 or more lipid abnormalities had significantly higher ORs for hypermethylation than those with no lipid abnormality (OR, 2.31; 95% CI, 1.11-4.82). CONCLUSION: LINE-1 DNA hypermethylation in leukocytes was associated with CVD risk profiles: high LDLC, high LDL/HDL ratio, and the degree of abnormal lipid metabolism.